Kinetics and mechanism of Ca2+ binding to arsenazo III and antipyrylazo III.

Equilibrium and temperature-jump spectrophotometric measurements were carried out on arsenazo III (Ar) and antipyrylazo III (Ap) in order to establish the kinetic reaction schemes for complexing of these dyes with Ca2+. The reaction media contained 30 mM Na2HPO4 as the buffer salt, at pH 7.4. Dependence of the relaxation rate of arsenazo III on dye and Ca2+ concentrations indicates the presence of both CaAr and CaAr2 complexes, with the CaAr2 form being responsible for the slow, 10-20 ms relaxation of this dye. For antipyrylazo III, the relaxation rate is much faster, less than 1 ms, and the complexing kinetics can be covered with only a CaAp complex. Unlike arsenazo III, antipyrylazo III binding with Ca2+ is rate-limited by a slow structural transition in the dye, taking antipyrylazo III from a low- to a high-affinity structure for Ca2+.